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Human brain specimen with glioblastoma multiforme.

Brain Cancer Cells Hide While Drugs Seek
Tumor cells temporarily lose mutation to evade drugs targeting mutation

A team of scientists, led by principal investigator Paul S. Mischel, MD, a member of the Ludwig Institute for Cancer Research and professor in the Department of Pathology at the University of California, San Diego School of Medicine, has found that brain cancer cells resist therapy by dialing down the gene mutation targeted by drugs, then re-amplify that growth-promoting mutation after therapy has stopped.

The findings are published in the December 5, 2013 online issue of Science.

“This discovery has considerable clinical implications because if cancer cells can evade therapy by a ‘hide-and-seek’ mechanism, then the current focus (of drug therapies) is unlikely to translate into better outcomes for patients,” said Mischel.

In recent years, new cancer therapies have emerged that target tell-tale gene mutations to identify specific cancer cells for destruction. Unfortunately, a variety of “resistance mechanisms” have also emerged, among them incomplete target suppression, second-site mutations and activation of alternative kinases or enzymes that maintain growth-promoting signals to the cancer itself. 

“Most research is aimed at developing better drugs or better drug combinations to suppress these downstream signals,” Mischel said. “However, one thing that has not been carefully considered is whether cancer cells can modulate the levels of – and thus their dependence on – the target of the drug, evade therapy, and then re-acquire the oncogene to promote tumor growth when the drug is withdrawn.”

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Omega-3 dietary supplements pass the blood-brain barrier

New research from Karolinska Institutet shows that omega-3 fatty acids in dietary supplements can cross the blood brain barrier in people with Alzheimer’s disease, affecting known markers for both the disease itself and inflammation. The findings are presented in the Journal of Internal Medicine, and strengthen the evidence that omega-3 may benefit certain forms of this seriously debilitating disease.

"Earlier population studies indicate that omega-3 can protect against Alzheimer’s disease, which makes it interesting to study the effects of dietary supplements containing this group of fatty acids in patients who have already developed the disease," says the study’s lead author Dr Yvonne Freund-Levi.

Omega-3 and other essential polyunsaturated fatty acids accumulate in the central nervous system (CNS) during gestation. It has been assumed that these acids are continually replaced throughout life, but little is known about how this occurs and whether changes in diet can affect the transport of important fatty acids across the blood-brain barrier. The blood-brain barrier serves to protect the brain from harmful chemicals existing naturally in the blood, but also blocks the delivery of drug substances to the brain.

Several diseases can affect the fatty acid profile of the CNS; in patients with Alzheimer’s disease, for example, previous research has observed lower than normal brain concentrations of docosahexaenoic acid (DHA), an omega-3 fatty acid.

In the present study, part of the larger OmegAD project, scientists examined whether omega-3 dietary supplements change the fatty acid profile of the CNS in patients with mild Alzheimer’s disease. Thirty-three patients participated in the study, 18 of whom received a daily omega-3 supplement and 15 a placebo for six months. The results show that the first group had higher levels of both DHA and eicosapentaenoic acid (EPA, another omega-3 fatty acid) in their cerebrospinal fluid (which surrounds the CNS) and blood. No such change was seen in the placebo group.

Moreover, they also found that levels of DHA correlated directly with the degree of change in Alzheimer’s disease and inflammatory markers in the cerebrospinal fluid. Researchers in the field have long been interested in this link between Alzheimer’s disease and inflammation, but attempts to treat the disease using traditional anti-inflammatory drugs have failed to produce any improvements in memory function.

"In animals, DHA dietary supplements can lead to an increase in DHA concentrations in the CNS," says Professor Jan Palmblad, who initiated the study. "Here we show that the same applies to humans, which suggests that omega-3 fatty acids in dietary supplements cross the blood-brain barrier. However, much work remains to be done before we know how these fatty acids can be used in the treatment of Alzheimer’s disease to halt memory loss."

Multi-dog study points to canine brain’s reward center

After capturing the first brain images of two alert, unrestrained dogs last year, researchers at Emory University have confirmed their methods and results by replicating them in an experiment involving 13 dogs.

The research, published by the Public Library of Science One (PLOS One), showed that most of the dogs had a positive response in the caudate region of the brain when given a hand signal indicating they would receive a food treat, as compared to a different hand signal for “no treat.”

“Our experiment last year was really a proof of concept, demonstrating that dogs could be trained to undergo successful functional Magnetic Resonance Imaging (fMRI),” says the lead researcher Gregory Berns, director of Emory’s Center for Neuropolicy. “Now we’ve shown that the initial study wasn’t a fluke: Canine fMRI is reliable and can be done with minimal stress to the dogs. We have laid the foundation for exploring the neural biology and cognitive processes of man’s best, and oldest, friend.”

Co-authors of the paper include Andrew Brooks, a post-doctoral fellow at the Center for Neuropolicy, and Mark Spivak, a dog trainer and the owner of Comprehensive Pet Therapy.

Both the initial experiment and the more recent one involved training the dogs to acclimatize to an fMRI machine. The task requires dogs to cooperatively enter the small enclosure of the fMRI scanner and remain completely motionless despite the noise and vibration of the machine.

Only those dogs that willingly cooperated were involved in the experiments. The canine subjects were given harmless fMRI brain scans while they watched a human giving hand signals that the dogs had been trained to understand. One signal indicated that the dog would receive a hot dog for a treat. The other hand signal meant that the dog would not receive a hot dog.

The most recent experiment involved the original two dogs, plus 11 additional ones, of varying breeds. Eight out of the 13 showed the positive caudate response for the hand signal indicating they were going to receive a hot dog.

The caudate sits above the brain stem in mammals and has the highest concentration of dopamine receptors, which are implicated in motivation and pleasure, among other neurological processes.

“We know that in humans, the caudate region is associated with decision-making, motivation and processing emotions,” Berns says.

As a point of reference, the researchers compared the results to a similar experiment Berns had led 10 years previously involving humans, in which the subjects pressed a button when a light appeared, to get a squirt of fruit juice.

Eleven of 17 humans involved in that experiment showed a positive response in the caudate region that was similar to the positive response of the dogs. “Our findings suggest that the caudate region of the canine brain behaves similarly to the caudate of the human brain, under similar circumstances,” Berns says.

Six of the dogs involved in the experiment had been specially bred and trained to assist disabled people as companion animals, and two of the dogs (including one of the service dogs) had worked as therapy dogs, used to help alleviate stress in people in hospitals or nursing homes. All of the service/therapy dogs showed a greater level of positive caudate activation for the hot dog signal, compared to the other dogs.

“We don’t know if the service dogs and therapy dogs showed this difference because of genetics, or because of the environment in which they were raised, but we hope to find out in future experiments,” Berns says. “This may be the first hint of how the brains of dogs with different temperaments and personalities differ.”

He adds: “I don’t think it was because they liked hot dogs more. I saw no evidence of that. None of the dogs turned down the hot dogs.”

One limitation of the experiments is the small number of subjects and the selectivity of the dogs involved, since only certain dogs can be trained to do the experiments, Berns says.

“We’re expanding our cohort to include more dogs and more breeds,” Berns says. “As the dogs get more accustomed to the process, we can conduct more complicated experiments.”

Plans call for comparing how the canine brain responds to hand signals coming from the dog’s owner, a stranger and a computer. Another experiment already under way is looking at the neural response of dogs when they are exposed to scents of members of their households, both humans and other dogs, and unfamiliar humans and dogs.

“Ultimately, our goal is to map out canine cognitive processes,” says Berns, who recently published a book entitled “How Dogs Love Us: A Neuroscientist and His Adopted Dog Decode the Canine Brain.”

Even in an increasingly technical era, the role of dogs has not diminished, Berns says. In addition to being popular pets, he notes that dogs are important in the U.S. military, in search-and-rescue missions, as assistants for the disabled and as therapeutic stress relievers for hospital patients and others.

“Dogs have been a part of human society for longer than any other animal,” Berns says. He cites a genetic analysis recently published in Science suggesting that the domestication of dogs goes back 18,000 to 32,000 years, preceding the development of agriculture some 10,000 years ago.

“Most neuroscience studies on animals are conducted to serve as models for human disease and brain functions,” Berns says. “We’re not studying canine cognition to serve as a model for humans, but what we learn about the dog brain may also help us understand more about how our own brains evolved.”

Study finds crocodiles are cleverer than previously thought

Turns out the crocodile can be a shrewd hunter himself. A University of Tennessee, Knoxville, researcher has found that some crocodiles use lures to hunt their prey.

Vladimir Dinets, a research assistant professor in the Department of Psychology, is the first to observe two crocodilian species—muggers and American alligators—using twigs and sticks to lure birds, particularly during nest-building time.

The research is published in the current edition of Ethology, Ecology and Evolution. Dinets’ research is the first report of tool use by any reptiles, and also the first known case of predators timing the use of lures to a seasonal behavior of the prey—nest-building.

Dinets first observed the behavior in 2007 when he spotted crocodiles lying in shallow water along the edge of a pond in India with small sticks or twigs positioned across their snouts. The behavior potentially fooled nest-building birds wading in the water for sticks into thinking the sticks were floating on the water. The crocodiles remained still for hours and if a bird neared the stick, they would lunge.

To see if the stick-displaying was a form of clever predation, Dinets and his colleagues performed systematic observations of the reptiles for one year at four sites in Louisiana, including two rookery and two nonrookery sites. A rookery is a bird breeding ground. The researchers observed a significant increase in alligators displaying sticks on their snouts from March to May, the time birds were building nests. Specifically, the reptiles in rookeries had sticks on their snouts during and after the nest-building season. At non-rookery sites, the reptiles used lures during the nest-building season.

"This study changes the way crocodiles have historically been viewed," said Dinets. "They are typically seen as lethargic, stupid and boring but now they are known to exhibit flexible multimodal signaling, advanced parental care and highly coordinated group hunting tactics."

The observations could mean the behavior is more widespread within the reptilian group and could also shed light on how crocodiles’ extinct relatives—dinosaurs—behaved.

"Our research provides a surprising insight into previously unrecognized complexity of extinct reptile behavior," said Dinets. "These discoveries are interesting not just because they show how easy it is to underestimate the intelligence of even relatively familiar animals, but also because crocodilians are a sister taxon of dinosaurs and flying reptiles."

Dinets collaborated with J.C and J.D. Brueggen from the St. Augustine Alligator Farm Zoological Park in St. Augustine, Fla. More of his crocodile research can be found in his book “Dragon Songs.”

How Our Nerves Keep Firing

University of Utah and German biologists discovered how nerve cells recycle tiny bubbles or “vesicles” that send chemical nerve signals from one cell to the next. The process is much faster and different than two previously proposed mechanisms for recycling the bubbles.

Researchers photographed mouse brain cells using an electron microscope after flash-freezing the cells in the act of firing nerve signals. That showed the tiny vesicles are recycled to form new bubbles only one-tenth of a second after they dump their cargo of neurotransmitters into the gap or “synapse” between two nerve cells or neurons.

“Without recycling these containers or ‘synaptic vesicles’ filled with neurotransmitters, you could move once and stop, think one thought and stop, take one step and stop, and speak one word and stop,” says University of Utah biologist Erik Jorgensen, senior author of the study in the Dec. 4 issue of the journal Nature.

“A fast nervous system allows you to think and move. Recycling synaptic vesicles allows your brain and muscles to keep working longer than a couple of seconds,” says Jorgensen, a distinguished professor of biology. “This process also may protect neurons from neurodegenerative diseases like Lou Gehrig’s disease and Alzheimer’s. So understanding the process may give us insights into treatments someday.”

A brain cell maintains a supply of 300 to 400 vesicles to send chemical nerve signals, using up to several hundred per second to release neurotransmitters, says the study’s first author, postdoctoral fellow Shigeki Watanabe.

Recycling vesicles is called “endocytosis.” Jorgensen and Watanabe named the process they observed “ultrafast endocytosis.” They showed it takes one-tenth of a second for a vesicle to be recycled, and such recycling occurs on the edge of “active zone” – the place on the end of the nerve cell where the vesicles first unload neurotransmitters into the synapse between brain cells.

“It’s like Whac-A-Mole: one vesicle goes down (fuses and unloads) and another pops up someplace else,” Jorgensen says.

Jorgensen believes ultrafast endocytosis is the most common way of recycling vesicles, but says the study doesn’t disprove two other, long-debated hypotheses:

– “Kiss-and-run endocytosis,” which supposedly takes one second, with a vesicle just “kissing” the inside of its nerve cell, dumping its neurotransmitters outside and “running” by detaching to reform a recycled vesicle in the same part of the active zone.

– Clathrin-mediated endocytosis,” which purportedly takes 20 seconds and occurs away from the active zone, at a point where a protein named clathrin assembles itself into a soccer-ball-shaped scaffold that forms a new vesicle or bubble.

Earlier this year, Jorgensen, Watanabe and colleagues published a related study in the journal eLife revealing that ultrafast endocytosis occurs in nematode worms. The new study of hippocampal brain cells from mice “tells us that mammals – and thus humans – do it the same way,” Jorgensen says. “The two papers together identify a process never previously seen – much faster than has been measured before.”

Jorgensen and Watanabe conducted the study with M. Wayne Davis, a University of Utah research assistant professor of biology; and technician Berit Söhl-Kielczynski and neuroscientists Christian Rosenmund, Benjamin Rost and Marcial Camacho-Pérez, all of Germany’s Charity University Medicine Berlin.

The study was funded by the National Institutes of Health, the European Research Council and the German Research Council. Jorgensen also is funded by his status as a Howard Hughes Medical Institute investigator and an Alexander von Humboldt Scholar.

Machine Gun Analogy for Vesicle Recycling

The process of a vesicle fusing to the nerve cell’s wall from the inside, then releasing neurotransmitters into the synapse is known as “exocytosis.” An analogy might be a bubble rising from boiling soup and releasing steam. The liquid part of the bubble fuses with the liquid in the soup, sooner or later to arise in another bubble.

The 2013 Nobel Prize in Physiology or Medicine went to three scientists who discovered key aspects of vesicle transport of cargo and exocytosis in nerve and other cells: which genes are required for vesicle transport, how vesicles deliver cargo to the correct locations, and how vesicles in brain cells release neurotransmitters to send a signal to the next brain neuron.

Jorgensen, Shigeki and colleagues studied the next step, endocytosis: how the membrane that forms vesicles (and nerve cell walls) is recycled to form new vesicles.

To illustrate the three possible mechanisms for recycling vesicles, Jorgensen compares vesicles with machine gun shells.

“You are fusing vesicles to the nerve cell membrane and expelling the neurotransmitter contents at extremely high rates,” he says. “The synapse will use up its ‘ammo’ very quickly at these rates, so the cell needs to refill the empty shells.”

Clathrin-mediated vesicle recycling is like “remaking the shell from scratch,” he says, while kiss-and-run endocytosis is like picking up every empty shell casing and refilling them one at a time.

“Ultrafast endocytosis allows the synapse to whip up all of the empty shells by the handful, fill them, and put them back in line at incredibly fast rates so the machine gun never runs out of ammo,” Jorgensen says.

Flash and Freeze for Nerve Cells in Action

Shigeki, Jorgensen and colleagues developed a method to photograph the tiny vesicles inside a nerve cell as the bubbles moved to the end of the cell, fused with the cell membrane, dumped their load of neurotransmitters into the gap or “synapse” between nerve cells, and then were recycled to reappear as new bubbles inside the nerve cell.

“We found a way to look at this process on a timescale that no one ever looked at before,” Watanabe says.

First, the researchers grew hundreds of brain cells from the mouse hippocampus – the often-studied part of the brain required for memory formation – on quarter-inch-wide sapphire disks placed in petri dishes with growth medium.

They added an algae gene to mouse brain cells that made the neurons produce an “ion channel” – basically a switch – that is stimulated by light instead of electricity. Then the brain cells were placed in a super-cold, high-pressure chamber, at 310 degrees below zero Fahrenheit and pressure 2,000 times greater than Earth’s atmosphere at sea level.

A wire cannot be routed into the chamber, which is why the cells were genetically programmed to be stimulated by light. The researchers flashed blue light on the mouse brain cells, making them “fire” neurotransmitter nerve signals. At the same time, the firing neurons were frozen with a blast of liquid nitrogen. To catch neurons in all stages of firing, the nerve cells were frozen at various times after the flash of blue light: 15, 30 and 100 milliseconds and one, three and 10 seconds.

“We built a new device to capture neurons performing fast behaviors,” Jorgensen says. “It stops all motion in the cell – even membranes in the act of fusing.

“We call it flash and freeze,” Watanabe says.

Next, the sapphire disks with neurons were put into liquid epoxy, which hardened and then were thin-sliced so the neurons could be photographed under an electron microscope. The ultrafast formation of recycled vesicles was visible.

“You see the outline of the membrane,” Jorgensen says. “You see the bubbles or vesicles in different stages of formation.”

Watanabe says about 3,000 mouse brain cell synapses were flashed, frozen and analyzed during the study. About 20 percent of the nerve cells had been fired and showed signs that nerve vesicles were being recycled.